Identification of differentially expressed genes in rat silicosis model by suppression subtractive hybridization analysis

Abstract

The critical molecular mechanism in the development of the pulmonary fibrosis remains unknown, leaving diagnosed patients with a poor prognosis. To isolate the genes specifically up-regulated in pulmonary fibrosis, we established a rat silicosis model 360 d after treatment with crystalline silica suspension. Radiographs of chests showed that some scattered high-density shadows appeared in the lung field. Typical microscopic fibrosing silicotic nodules formed in the lung, alveolar epithelial cells and bronchial epithelial cells, particularly around the partial fibrosing silicotic nodules; some of them showed atypical hyperplasia that suggested a correlation between silicosis and lung cancer. Suppression subtractive hybridization analysis was performed to compare gene expression in lung tissue with silicosis and normal lung tissue. Reverse transcription-polymerase chain reaction showed that the expressions of seven novel cDNA sequences identified by suppression subtractive hybridization in lung tissue with silicosis differed from normal lung tissue. Bioinformatics analysis showed that 47 positive clones represented 35 genes containing two putative proteins and four predicted similar proteins. The analysis also showed that some screened genes in silicosis, such as prolyl 4-hydroxylases, actin-related protein-2/3 complex and acidic mammalian chitinase, have not been previously reported. These genes may provide new clues for investigating the molecular mechanisms in the development of pulmonary fibrosis.

Lung Fibrosis: Drug Screening and Disease Biomarker Identification with a Lung Slice Culture Model and Subtracted cDNA Library

“…Suppressive subtractive hybridisation (SSH) is a commonly used method (14-16) that has been successfully applied in studies on tumours, developmental biology and immune regulation (17)(18)(19). Recently, SSH has been employed to identify genes related to pulmonary fibrosis (20,21). Asymmetric subtractive hybridisation (ASH)-subtracted cDNA library construction technology, developed by Bio S&T, is a subtractive hybridisation protocol that uses single-stranded DNA in subtractive hybridisation, which results in much higher efficiency in the enrichment of differentially-expressed genes, in contrast to the double-stranded DNA approach of the SSH method.…”